yitJ

ABSTRACT

The invention provides yitJ polypeptides and DNA (RNA) encoding yitJ polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing yitJ polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/027,032, filed Sep. 24, 1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the Cystathionine gamma-lyase family, hereinafterreferred to as "yitJ".

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past 20 years. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This has created a demand for both newanti-microbial agents and diagnostic tests for this organism.

Cystathionine gamma-lyase is an enzyme involve in the biosynthesis ofcysteine and alpha-ketobutyrate from cystathionine. The cystathionine isitself formed from S-adenosyl methionine and serine. Yit J is a paralogof the gene encoding the gammalyase and encodes a protein involved incatalysing a similar reaction.

Substantial effort has been invested this century in the successfuldiscovery and development of antibacterials. Paradoxically, althoughantibacterials are devised to eradicate infection in mammals we knowalmost nothing of the physiology of bacterial pathogens in infectivesituations in the host. Using sequences from the Staphylococcus aureuschromosome we have developed an RT-PCR based procedure which allows usto identify those bacterial genes transcribed at any stage of infectionand also from different niches of infection. The derivation of suchinformation is a critical first step in understanding the globalresponse of the bacterial gene complement to the host environment. Fromthe knowledge of bacterial genes both of known and unknown functionwhich are widely transcribed in the host it is possible to attempt toascertain by database searching those which are present only in theeubacteria. Further prioritisation of such genes by consideration of thelikely role of their products towards the maintenance of infection andthe facility of setting up a screen for inhibitors of the biochemicalfunction indicated by their homology to characterised genes allows thecompilation of a shortlist for gene essentiality studies using geneticdeletion or controlled regulation techniques. The proteins expressed bygenes shown to be necessary for growth in vitro or in pathogenesis inanimal models provide novel targets for antibacterial screening to findagents which are broadly inhibitory towards pathogenesis. This inventionprovides S.aureus WCUH 29 polynucleotides which are transcribed ininfected tissue, in particular in both acute and chronic infections.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown yitJ EMBL: locus BSY09476, accession Y09476! protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel yitJ polypeptides by homology between the amino acidsequence set out in Table 1 SEQ ID NO: 2! and a known amino acidsequence or sequences of other proteins such as yitJ EMBL: locusBSY09476, accession Y09476! protein.

It is a further object of the invention to provide polynucleotides thatencode yitJ polypeptides, particularly polynucleotides that encode thepolypeptide herein designated yitJ.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding yitJ polypeptides comprisingthe sequence set out in Table 1 SEQ ID NO: 1! which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel yitJ protein from Staphylococcus aureus comprising the amino acidsequence of Table 1 SEQ ID NO:2!, or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in the depositedstrain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding yitJ, particularly Staphylococcus aureus yitJ,including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of yitJ and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as yitJ as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of yitJ polypeptide encoded by naturally occurring alleles ofthe yitJ gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned yitJ polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing yitJexpression, treating disease, for example, disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), assaying genetic variation, and administeringa yitJ polypeptide or polynucleotide to an organism to raise animmunological response against a bacteria, especially a Staphylococcusaureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to yitJ polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against yitJ polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor pilynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided yitJ agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a yitJ polynucleotide or a yitJ polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel yitJ polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel yitJ of Staphylococcusaureus, which is related by amino acid sequence homology to yitJ EMBL:locus BSY09476, accession Y09476! polypeptide. The invention relatesespecially to yitJ having the nucleotide and amino acid sequences setout in Table 1 SEQ ID NO: 1! and Table 1 SEQ ID NO: 2! respectively, andto the yitJ nucleotide sequences of the DNA in the deposited strain andamino acid sequences encoded thereby.

                                      TABLE 1    __________________________________________________________________________    YitJ Polynucleotide and Polypeptide Sequences    __________________________________________________________________________    (A)      Sequences from Staphylococcus aureus yitJ polynucleotide sequence  SEQ      ID NO: 1!.    5'-1  ATGAGTCAAT                  TCCTCACACA                          ATTGAAAGAT                                   AATGTTTTAG                                           TAGCTGATGG    51    CGCTATTGGA                  ACCATTTTAT                          ACTCTGAAGG                                   ATTAGACACC                                           TGTCCAGAAG    101   CATATAATCT                  TAGCCATCCA                          GATAAAGTTG                                   AACGCATCCA                                           TCGTTCATAT    151   ATTGAAGCCG                  GTGCTGATGT                          CATTCAAACC                                   AATACTTATG                                           GTGCAAATTT    201   TGAAAAGTTA                  AAACGATTCG                          GTCTTGAAGA                                   TAAAGTTAAA                                           GCAATACATC    251   AAGCCGCCGT                  TCGCATCGCA                          AAAAAAGCAG                                   CAAATAAAGA                                           TACGTATATA    301   TTAGGCACAG                  TTGGTGGGTT                          TAGAGGTATC                                   AAACAAGAGG                                           ATATCAGCTT    351   ACAAACTATT                  CTTTATCATA                          CTGAAATTCA                                   AATAGACACC                                           TTAATTGAAG    401   AAGGCGTTGA                  CGCGCTACTT                          TTCGAAACGT                                   ATTACGACCT                                           AGAAGAGTTA    451   ACAAATGTCA                  TTTCACGAAC                          GAGAAAGAAA                                   TACGACATTC                                           CAATCATTGC    501   TCAATTAACC                  GCTTCAAACA                          CAAATTACTT                                   AGTTAATGGT                                           CAGGCAATCA    551   ATGAAGGATT                  AAAACAACTC                          GTTCAATGTG                                   GTGCAAACAT                                           CGTGGGACTC    601   AATTGTCATC                  ATGGTCCGCA                          CCATATGCAA                                   GAGTCTTTCA                                           CACATATTGA    651   ATTACCAGAG                  CACGCATTCT                          TATCTTGTTA                                   TCCAAATGCC                                           AGCTTATTAG    701   ATATTGAAAA                  TAGTGAATTT                          AAGTATAGTG                                   ACAATGCACA                                           ATATTTCGGT    751   CAAGTTGCTC                  AAAATCTAAT                          TCGCGAAGGT                                   GTTCGTTTAA                                           TTGGTGGTTG    801   CTGTGGTACA                  ACGCCAGAGC                          ACATCAAATT                                   TATTAAAGAA                                           TCTATTCAGA    851   CACTTAAACC                  TGTTAATGAC                          AAAAAAGTGA                                   TTCCGATACC                                           AACGAAAGCA    901   CTTTTCAATC                  CATCTCAAAA                          TAAAGTTAGA                                   CAATCATTAA                                           CATCTAAGGT    951   TCAAGAACGT                  CCAACCGTTA                          TTATCGAATT                                   GGATACACCG                                           AAACATTTAG    1001  ACACGGATAG                  ATTTTTTGAA                          AATATCGCTA                                   AACTTGATAA                                           AGCTAATGTA    1051  GATGCGGTAA                  CACTCGCAGA                          TAATTCATTG                                   GCAACTGTCA                                           GAATTAGCAA    1101  TATTGCTGCT                  GCTAGCTTAA                          TTAAGCAATA                                   TTACAATATT                                           GAACCACTCG    1151  TACATATTAC                  ATGTCGAGAC                          CGAAACTTAA                                   TCGGCTTGCA                                           GTCCCATTTA    1201  CTTGGATTAT                  CGCTCATTGG                          CGTTAACGAA                                   ATATTAGCCA                                           TAACTGGTGA    1251  TCCTTCAAAA                  GTTGGTCACT                          TACCAGGTGC                                   AACCAATGTC                                           TATGATGTTA    1301  ATTCTAAAGG                  ATTAACTGAA                          CTCGCTCTAA                                   GATTTAATCA                                           AGGTATTAAC    1351  ACTGACGGTG                  ATGCGCTGAA                          GAAACGTACA                                   CACTTCAACA                                           TCGCTGGCGC    1401  CTTTAACCCT                  AATGTTCGTA                          AATTAGATGG                                   TGCCGTCAAA                                           AGATTAGAGA    1451  AAAAGATAGA                  AAGCGGAATG                          TCTTATTTTA                                   TAACACAACC                                           CGTCTACAGC    1501  AAAGAGAAAA                  TCATTGAAAT                          TTACCATGCC                                   ACTAAGCACT                                           TGAACAAACC    1551  ATTTTTCATA                  GGCATTATGC                          CTATCGCAAG                                   TTACAAAAAC                                           GCACTCTTTT    1601  TGCATAATGA                  AGTGCCAGGA                          ATCAAGATGT                                   CAGATGAAAT                                           TTTACAACAA    1651  TTTGAAGCAG                  TTAAAGATGA                          TAAAGCCAAA                                   ACACGAGAAC                                           TAAGTCTTAA    1701  GCTTTCAAAG                  GATTTAATCG                          ATACTGTTCA                                   TGAATATTTT                                           AATGGTTTAT    1751  ACATTATCAC                  ACCGTTTCAA                          AATGTCGAAG                                   ATTCATTAGA                                           ACTTGCAGCA    1801  TACTCAAAAT                  CTATTACTGC                          TCACAAGGAG                                   GCAATATTAT                                           GA    (B)      yitJ polypeptide sequence deduced from the polynucleotide sequence in      this table  SEQ ID NO: 2!.    NH.sub.2 -1          MSQFLTQLKD                  NVLVADGAIG                          TILYSEGLDT                                   CPEAYNLSHP                                           DKVERIHRSY    51    IEAGADVIQT                  NTYGANFEKL                          KRFGLEDKVK                                   AIHQAAVRIA                                           KKAANKDTYI    101   LGTVGGFRGI                  KQEDISLQTI                          LYHTEIQIDT                                   LIEEGVDALL                                           FETYYDLEEL    151   TNVISRTRKK                  YDIPIIAQLT                          ASNTNYLVNG                                   QAINEGLKQL                                           VQCGANIVGL    201   NCHHGPHHMQ                  ESFTHIELPE                          HAFLSCYPNA                                   SLLDIENSEF                                           KYSDNAQYFG    251   QVAQNLIREG                  VRLIGGCCGT                          TPEHIKFIKE                                   SIQTLKPVND                                           KKVIPIPTKA    301   LFNPSQNKVR                  QSLTSKVQER                          PTVIIELDTP                                   KHLDTDRFFE                                           NIAKLDKANV    351   DAVTLADNSL                  ATVRISNIAA                          ASLIKQYYNI                                   EPLVHITCRD                                           RNLIGLQSHL    401   LGLSLIGVNE                  ILAITGDPSK                          VGHLPGATNV                                   YDVNSKGLTE                                           LALRFNQGIN    451   TDGDALKKRT                  HFNIAGAFNP                          NVRKLDGAVK                                   RLEKKIESGM                                           SYFITQPVYS    501   KEKIIEIYHA                  TKHLNKPFFI                          GIMPIASYKN                                   ALFLHNEVPG                                           IKMSDEILQQ    551   FEAVKDDKAK                  TRELSLKLSK                          DLIDTVHEYF                                   NGLYIITPFQ                                           NVEDSLELAA    601   YSKSITAHKE                  AIL--COOH    (C)      Polynucleotide sequence embodiments  SEQ ID NO: 1!.    X--(R.sub.1).sub.n -1          ATGAGTCAAT                  TCCTCACACA                          ATTGAAAGAT                                   AATGTTTTAG                                           TAGCTGATGG    51    CGCTATTGGA                  ACCATTTTAT                          ACTCTGAAGG                                   ATTAGACACC                                           TGTCCAGAAG    101   CATATAATCT                  TAGCCATCCA                          GATAAAGTTG                                   AACGCATCCA                                           TCGTTCATAT    151   ATTGAAGCCG                  GTGCTGATGT                          CATTCAAACC                                   AATACTTATG                                           GTGCAAATTT    201   TGAAAAGTTA                  AAACGATTCG                          GTCTTGAAGA                                   TAAAGTTAAA                                           GCAATACATC    251   AAGCCGCCGT                  TCGCATCGCA                          AAAAAAGCAG                                   CAAATAAAGA                                           TACGTATATA    301   TTAGGCACAG                  TTGGTGGGTT                          TAGAGGTATC                                   AAACAAGAGG                                           ATATCAGCTT    351   ACAAACTATT                  CTTTATCATA                          CTGAAATTCA                                   AATAGACACC                                           TTAATTGAAG    401   AAGGCGTTGA                  CGCGCTACTT                          TTCGAAACGT                                   ATTACGACCT                                           AGAAGAGTTA    451   ACAAATGTCA                  TTTCACGAAC                          GAGAAAGAAA                                   TACGACATTC                                           CAATCATTGC    501   TCAATTAACC                  GCTTCAAACA                          CAAATTACTT                                   AGTTAATGGT                                           CAGGCAATCA    551   ATGAAGGATT                  AAAACAACTC                          GTTCAATGTG                                   GTGCAAACAT                                           CGTGGGACTC    601   AATTGTCATC                  ATGGTCCGCA                          CCATATGCAA                                   GAGTCTTTCA                                           CACATATTGA    651   ATTACCAGAG                  CACGCATTCT                          TATCTTGTTA                                   TCCAAATGCC                                           AGCTTATTAG    701   ATATTGAAAA                  TAGTGAATTT                          AAGTATAGTG                                   ACAATGCACA                                           ATATTTCGGT    751   CAAGTTGCTC                  AAAATCTAAT                          TCGCGAAGGT                                   GTTCGTTTAA                                           TTGGTGGTTG    801   CTGTGGTACA                  ACGCCAGAGC                          ACATCAAATT                                   TATTAAAGAA                                           TCTATTCAGA    851   CACTTAAACC                  TGTTAATGAC                          AAAAAAGTGA                                   TTCCGATACC                                           AACGAAAGCA    901   CTTTTCAATC                  CATCTCAAAA                          TAAAGTTAGA                                   CAATCATTAA                                           CATCTAAGGT    951   TCAAGAACGT                  CCAACCGTTA                          TTATCGAATT                                   GGATACACCG                                           AAACATTTAG    1001  ACACGGATAG                  ATTTTTTGAA                          AATATCGCTA                                   AACTTGATAA                                           AGCTAATGTA    1051  GATGCGGTAA                  CACTCGCAGA                          TAATTCATTG                                   GCAACTGTCA                                           GAATTAGCAA    1101  TATTGCTGCT                  GCTAGCTTAA                          TTAAGCAATA                                   TTACAATATT                                           GAACCACTCG    1151  TACATATTAC                  ATGTCGAGAC                          CGAAACTTAA                                   TCGGCTTGCA                                           GTCCCATTTA    1201  CTTGGATTAT                  CGCTCATTGG                          CGTTAACGAA                                   ATATTAGCCA                                           TAACTGGTGA    1251  TCCTTCAAAA                  GTTGGTCACT                          TACCAGGTGC                                   AACCAATGTC                                           TATGATGTTA    1301  ATTCTAAAGG                  ATTAACTGAA                          CTCGCTCTAA                                   GATTTAATCA                                           AGGTATTAAC    1351  ACTGACGGTG                  ATGCGCTGAA                          GAAACGTACA                                   CACTTCAACA                                           TCGCTGGCGC    1401  CTTTAACCCT                  AATGTTCGTA                          AATTAGATGG                                   TGCCGTCAAA                                           AGATTAGAGA    1451  AAAAGATAGA                  AAGCGGAATG                          TCTTATTTTA                                   TAACACAACC                                           CGTGTACAGC    1501  AAAGAGAAAA                  TCATTGAAAT                          TTACCATGCC                                   ACTAAGCACT                                           TGAACAAACC    1551  ATTTTTCATA                  GGCATTATGC                          CTATCGCAAG                                   TTACAAAAAC                                           GCACTCTTTT    1601  TGCATAATGA                  AGTGCCAGGA                          ATCAAGATGT                                   CAGATGAAAT                                           TTTACAACAA    1651  TTTGAAGCAG                  TTAAAGATGA                          TAAAGCCAAA                                   ACACGAGAAC                                           TAAGTCTTAA    1701  GCTTTCAAAG                  GATTTAATCG                          ATACTGTTCA                                   TGAATATTTT                                           AATGGTTTAT    1751  ACATTATCAC                  ACCGTTTCAA                          AATGTCGAAG                                   ATTCATTAGA                                           ACTTGCAGCA    1801  TACTCAAAAT                  CTATTACTGC                          TCACAAGGAG                                   GCAATATTAT                                           GA--(R.sub.2).sub.n -Y    (D)      Polypeptide sequence embodiments  SEQ ID NO: 2!.    X--(R.sub.1).sub.n -1          MSQFLTQLKD                  NVLVADGAIG                          TILYSEGLDT                                   CPEAYNLSHP                                           DKVERIHRSY    51    IEAGADVIQT                  NTYGANFEKL                          KRFGLEDKVK                                   AIHQAAVRIA                                           KKAANKDTYI    101   LGTVGGFRGI                  KQEDISLQTI                          LYHTEIQIDT                                   LIEEGVDALL                                           FETYYDLEEL    151   TNVISRTRKK                  YDIPIIAQLT                          ASNTNYLVNG                                   QAINEGLKQL                                           VQCGANIVGL    201   NCHHGPHHMQ                  ESFTHIELPE                          HAFLSCYPNA                                   SLLDIENSEF                                           KYSDNAQYFG    251   QVAQNLIREG                  VRLIGGCCGT                          TPEHIKFIKE                                   SIQTLKPVND                                           KKVIPIPTKA    301   LFNPSQNKVR                  QSLTSKVQER                          PTVIIELDTP                                   KHLDTDRFFE                                           NIAKLDKANV    351   DAVTLADNSL                  ATVRISNIAA                          ASLIKQYYNI                                   EPLVHITCRD                                           RNLIGLQSHL    401   LGLSLIGVNE                  ILAITGDPSK                          VGHLPGATNV                                   YDVNSKGLTE                                           LALRFNQGIN    451   TDGDALKKRT                  HFNIAGAFNP                          NVRKLDGAVK                                   RLEKKIESGM                                           SYFITQPVYS    501   KEKIIEIYHA                  TKHLNKPFFI                          GIMPIASYKN                                   ALFLHNEVPG                                           IKMSDEILQQ    551   FEAVKDDKAK                  TRELSLKLSK                          DLIDTVHEYF                                   NGLYIITPFQ                                           NVEDSLELAA    601   YSKSITAHKE                  AIL--(R.sub.2).sub.n -Y    (E)      Sequences from Staphlococcus aureus yitJ polynucleotide ORF sequence       SEQ ID NO: 3!.    5'-1  ATGAGTCAAT                  TCCTCACACA                          ATTGAAAGAT                                   AATGTTTTAG                                           TAGTGATGG    51    CGCTATTGGA                  ACCATTTTAT                          ACTCTGAAGG                                   ATTAGACACC                                           TGTCCAGAAG    101   CATATAATCT                  TAGCCATCCA                          GATAAAGTTG                                   AACGCATCCA                                           TCGTTCATAT    151   ATTGAAGCCG                  GTGCTGATGT                          CATTCAAACC                                   AATACTTATG                                           GTGCAAATTT    201   TGAAAAGTTA                  AAACGATTCG                          GTCTTGAAGA                                   TAAAGTTAAA                                           GCAATACATC    251   AAGCCGCCGT                  TCGCATCGCA                          AAAAAAGCAG                                   CAAATAAAGA                                           TACGTATATA    301   TTAGGCACAG                  TTGGTGGGTT                          TAGAGGTATC                                   AAACAAGAGG                                           ATATCAGCTT    351   ACAAACTATT                  CTTTATCATA                          CTGAAATTCA                                   AATAGACACC                                           TTAATTGAAG    401   AAGGCGTTGA                  CGCGCTACTT                          TTCGAAACGT                                   ATTACGACCT                                           AGAAGAGTTA    451   ACAAATGTCA                  TTTCACGAAC                          GAGAAAGAAA                                   TACGACATTC                                           CAATCATTGC    501   TCAATTAACC                  GCTTCAAACA                          CAAATTACTT                                   AGTTAATGGT                                           CAGGCAATCA    551   ATGAAGGATT                  AAAACAACTC                          GTTCAATGTG                                   GTGCAAACAT                                           CGTGGGACTC    601   AATTGTCATC                  ATGGTCCGCA                          CCATATGCAA                                   GAGTCTTTCA                                           CACATATTGA    651   ATTACCAGAG                  CACGCATTCT                          TATCTTGTTA                                   TCCAAATGCC                                           AGCTTATTAG    701   ATATTGAAAA                  TAGTGAATTT                          AAGTATAGTG                                   ACAATGCACA                                           ATATTTCGGT    751   CAAGTTGCTC                  AAAATCTAAT                          TCGCGAAGGT                                   GTTCGTTTAA                                           TTGGTGGTTG    801   CTGTGGTACA                  ACGCCAGAGC                          ACATCAAATT                                   TATTAAAGAA                                           TCTATTCAGA    851   CACTTAAACC                  TGTTAATGAC                          AAAAAAGTGA                                   TTCCGATACC                                           AACGAAAGCA    901   CTTTTCAATC                  CATCTCAAAA                          TAAAGTTAGA                                   CAATCATTAA                                           CATCTAAGGT    951   TCAAGAACGT                  CCAACCGTTA                          TTATCGAATT                                   GGATACACCG                                           AAACATTTAG    1001  ACACGGATAG                  ATTTTTTGAA                          AATATCGCTA                                   AACTTGATAA                                           AGCTAATGTA    1051  GATGCGGTAA                  CACTCGCAGA                          TAATTCATTG                                   GCAACTGTCA                                           GAATTAGCAA    1101  TATTGCTGCT                  GCTAGCTTAA                          TTAAGCAATA                                   TTACAATATT                                           GAACCACTCG    1151  TACATATTAC                  ATGTCGAGAC                          CGAAACTTAA                                   TCGGCTTGCA                                           GTCCCATTTA    1201  CTTGGATTAT                  CGCTCATTGG                          CGTTAACGAA                                   ATATTAGCCA                                           TAACTGGTGA    1251  TCCTTCAAAA                  GTTGGTCACT                          TACCAGGTGC                                   AACCAATGTC                                           TATGATGTTA    1301  ATTCTAAAGG                  ATTAACTGAA                          CTCGCTCTAA                                   GATTTAATCA                                           AGGTATTAAC    1351  ACTGACGGTG                  ATGCGCTGAA                          GAAACGTACA                                   CACTTCAACA                                           TCGCTGGCGC    1401  CTTTAACCCT                  AATGTTCGTA                          AATTAGATGG                                   TGCCGTCAAA                                           AGATTAGAGA    1451  AAAAGATAGA                  AAGCGGAATG                          TCTTATTTTA                                   TAACACAACC                                           CGTGTACAGC    1501  AAAGAGAAAA                  TCATTGAAAT                          TTACCATGCC                                   ACTAAGCACT                                           TGAACAAACC    1551  ATTTTTCATA                  GGCATTATGC                          CTATCGCAAG                                   TTACAAAAAC                                           GCACTCTTTT    1601  TGCATAATGA                  AGTGCCAGGA                          ATCAAGATGT                                   CAGATGAAAT                                           TTTACAACAA    1651  TTTGAAGCAG                  TTAAAGATGA                          TAAAGCCAAA                                   ACACGAGAAC                                           TAAGTCTTAA    1701  GCTTTCAAAG                  GATTTAATCG                          ATACTGTTCA                                   TGAATATTTT                                           AATGGTTTAT    1751  ACATTATCAC                  ACCGTTTCAA                          AATGTCGAAG                                   ATTCATTAGA                                           ACTTGCAGCA    1801  TACTCAAAAT                  CTATTACTGC                          TCACAAGGAG                                   GCAATATTA-3'    (F)      YitJ polypeptide sequence deduced from the polynucleotide ORF sequence      in this table       SEQ ID NO: 4!.    NH.sub.2 -1          MSQFLTQLKD                  NVLVADGAIG                          TILYSEGLDT                                   CPEAYNLSHP                                           DKVERIHRSY    51    IEAGADVIQT                  NTYGANFEKL                          KRFGLEDKVK                                   AIHQAAVRIA                                           KKAANKDTYI    101   LGTVGGFRGI                  KQEDISLQTI                          LYHTEIQIDT                                   LIEEGVDALL                                           FETYYDLEEL    151   TNVISRTRKK                  YDIPIIAQLT                          ASNTNYLVNG                                   QAINEGLKQL                                           VQCGANIVGL    201   NCHHGPHHMQ                  ESFTHIELPE                          HAFLSCYPNA                                   SLLDIENSEF                                           KYSDNAQYFG    251   QVAQNLIREG                  VRLIGGCCGT                          TPEHIKFIKE                                   SIQTLKPVND                                           KKVIPIPTKA    301   LFNPSQNKVR                  QSLTSKVQER                          PTVIIELDTP                                   KHLDTDRFFE                                           NIAKLDKANV    351   DAVTLADNSL                  ATVRISNIAA                          ASLIKQYYNI                                   EPLVHITCRD                                           RNLIGLQSHL    401   LGLSLIGVNE                  ILAITGDPSK                          VGHLPGATNV                                   YDVNSKGLTE                                           LALRFNQGIN    451   TDGDALKKRT                  HFNIAGAFNP                          NVRKLDGAVK                                   RLEKKIESGM                                           SYFITQPVYS    501   KEKIIEIYHA                  TKHLNKPFFI                          GIMPIASYKN                                   ALFLHNEVPG                                           IKMSDEILQQ    551   FEAVKDDKAK                  TRELSLKLSK                          DLIDTVHEYF                                   NGLYIITPFQ                                           NVEDSLELAA    601   YSKSITAHKE                  AIL--COOH    __________________________________________________________________________

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11 Sep. 1995 and assigned NCIMB Deposit No. 40771, and isreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as "thedeposited strain" or as "the DNA of the deposited strain." The depositedstrain contains the fall length yitJ gene. The sequence of thepolynucleotides contained in the deposited strain, as well as the aminoacid sequence of the polypeptide encoded thereby, are controlling in theevent of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1 SEQID NO:2! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofyitJ, and also those which have at least 70% identity to a polypeptideof Table 1 SEQ ID NOS:2 and 4! or the relevant portion, preferably atleast 80% identity to a polypeptide of Table 1 SEQ ID NOS:2 and 4!, andmore preferably at least 90% similarity (more preferably at least 90%identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) SEQ ID NO:2! wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with yitJ polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 SEQ ID NOS:2 and 4!, orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or fumctional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of yitJ, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the yitJ polypeptide havinga deduced amino acid sequence of Table 1 SEQ ID NOS:2 and 4! andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 SEQ ID NOS:1 and 3!, a polynucleotide of theinvention encoding yitJ polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a fall lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 SEQ ID NOS:1 and 3!,typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989) (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 SEQ ID NO:1! was discovered in a DNA library derived fromStaphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 SEQ ID NOS:1! contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 SEQ ID NO:2! with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO: 1,between nucleotide number 1 through number 1839 encodes the polypeptideof SEQ ID NO:2. The stop codon begins at nucleotide number 1840 of SEQID NO:1.

Yitj of the invention is structurally related to other proteins of theCystathionine gamma-lyase family, as shown by the results of sequencingthe DNA encoding yitJ of the deposited strain. The protein exhibitsgreatest homology to yitJ EMBL: locus BSY09476, accession Y09476!protein among known proteins. YitJ of Table 1 SEQ ID NO:2! has about 49%identity over its entire length and about 70% similarity over its entirelength with the amino acid sequence of yitJ EMBL: locus BSY09476,accession Y09476! polypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 SEQ ID NO:1!. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to 1839 or 1842 set forth in SEQ ID NO:1 ofTable 1 which encode the yitJ polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) SEQ ID NO:1! wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus yitJ havingthe amino acid sequence set out in Table 1 SEQ ID NO:2!. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 SEQ ID NO:2!. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingyitJ variants, that have the amino acid sequence of yitJ polypeptide ofTable 1 SEQ ID NO:2! in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof yitJ.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding yitJ polypeptide having an amino acid sequence set out in Table1 SEQ ID NOS:2 and 4!, and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding yitJ polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 SEQ ID NO:1!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the hybridization support in 0.1× SSC atabout 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions witha probe having the sequence of said polynucleotide sequence set forth inSEQ ID NO:1 or a fragment thereof; and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding yitJ and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the yitJ gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the yitJ gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the yitJ polynucleotides ofthe invention for use as diagnostic reagents. Detection of yitJ in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans, infectedwith an organism comprising the yitJ gene may be detected at the nucleicacid level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled yitJ polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g. Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g. Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding yitJ can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2.

                  TABLE 2    ______________________________________    Primers for ampliflcation of yitJ polynucleotides    SEQ ID NO   PRIMER SEQUENCE    ______________________________________    5           5'-CCTCACACAA TTGAAAGATA ATG-3'    6           5'-GTGAAAGACT CTTGCATATG G-3'    ______________________________________

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying yitJ DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 SEQ ID NO: 1!. Increased or decreasedexpression of yitJ polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of yitJ protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a yitJprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-yitJ or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope-termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against yitJ- polypeptide may be employedto treat infections, particularly bacterial infections and especiallydisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of yitJpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising yitJ polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a yitJ agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the yitJ polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of yitJ polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in yitJ polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for yitJ antagonists is a competitive assaythat combines yitj and a potential antagonist with yitJ-bindingmolecules, recombinant yitJ binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. YitJ can be labeled, such as byradioactivity or a colorimetric compound, such that the number of yitJmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing yitJ-induced activities, thereby preventing the action of yitJby excluding yitJ from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of yitJ.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgamo or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock yitJ protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial yitJ proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with yitJ, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of yitJ, or afragment or a variant thereof, for expressing yitJ, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a yitJ or protein coded therefrom,wherein the composition comprises a recombinant yitJ or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid yitJ or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A yitJ polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain yitJprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared byroutine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of thefollowing two methods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 YitJ Characterization

The determination of expression during infection of a gene fromStaphylococcus aureus entails the following procedures:

Necrotic fatty tissue from a 72 hour groin infection or an excisedkidney from an 8 day chronic kidney infection of Staphylococcus aureusWCUH29 in the mouse is efficiently disrupted and processed in thepresence of chaotropic agents and RNAase inhibitor to provide a mixtureof animal and bacterial RNA. The optimal conditions for disruption andprocessing to give stable preparations and high yields of bacterial RNAare followed by the use of hybridisation to a radiolabelledoligonucleotide specific to Staphylococcus aureus 16S RNA on Northernblots. The RNAase free, DNAase free, DNA and protein free preparationsof RNA obtained are suitable for Reverse Transcription PCR (RT-PCR)using unique primer pairs designed from the sequence of each gene ofStaphylococcus aureus WCUH29.

a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amouse animal model of infection (groin):

10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded withisolated, individual colonies of Staphylococcus aureus WCUH29 from anagar culture plate. The cultures are incubated aerobically (staticculture) at 37° C. for 16-20 hours. 4 week old mice (female,18 g-22 g,strain MF1) are each infected by subcutaneous injection of 0.5 ml ofthis broth culture of Staphylococcus aureus WCUH29 (diluted in broth toapproximately 10⁸ cfu/ml.) into the anterior, right lower quadrant(groin area). Mice are monitored regularly during the first 24 hoursafter infection, then daily until termination of study. Animals withsigns of systemic infection, i.e. lethargy, ruffled appearance,isolation from group, are monitored closely and if signs progress tomoribundancy, the animal are culled immediately.

Visible external signs of lesion development are seen 24-48 hours afterinfection. Examination of the abdomen of the animal shows the raisedoutline of the abscess beneath the skin. The localised lesion remains inthe right lower quadrant, but may occasionally spread to the left lowerquadrant, and superiorly to the thorax. On occasions, the abscess mayrupture through the overlying skin layers. In such cases, the affectedanimal is culled immediately and the tissues sampled, if possible.Failure to cull the animal may result in the necrotic skin tissueoverlying the abscess being sloughed off, exposing the abdominal musclewall.

Approximately 96 hours after infection, animals are killed using carbondioxide asphyxiation. To minimise delay between death and tissueprocessing/storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care is takennot to penetrate the abdominal wall. Holding the skin flap with forceps,the skin is gently pulled way from the abdomen. The exposed abscess,which covers the peritoneal wall but generally does not penetrate themuscle sheet completely, is excised, taking care not to puncture theviscera

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

b) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amurine model of hematogenous pyelonephritis:

Overnight cultures of S. aureus WCUH29 are started from single coloniesin 5 ml of tryptic soy broth (TSB) and grown at 37° C. with shaking. Thecultures are then washed twice in sterile phosphate-buffered saline(PBS) and diluted to an A600=0.3. Male CD-1 mice (18-20 g) were infectedwith 0.2 ml of this suspension by tail vein inoculation using a 30 gaugeneedle attached to a tuberculin syringe. Each mouse receivesapproximately 4×10⁷ bacteria in this fashion. Mice are monitored dailyfor signs of illness, and usually within 48 hours show signs oflethargy, ruffled fur, sluggishness; animals which appear moribund areeuthanized prior to the end of the experiment.

All animals are euthanized via carbon dioxide overdose seven dayspost-infection. The animal is placed on its back and swabbed withethanol, and then with RNAZap, and instruments are swabbed as well. Theabdominal cavity is opened and the kidneys aseptically removed, cut intofour pieces, and placed in cryovials which are immediately frozen inliquid nitrogen.

c) Isolation of Staphylococcus aureus WCURH29 RNA from infected tissuesamples:

4-6 infected tissue samples (each approx 0.5-0.7 g) in 2 ml screw-captubes are removed from -80° C. storage into a dry ice ethanol bath. In amicrobiological safety cabinet the samples are disrupted individuallywhilst the remaining samples are kept cold in the dry ice ethanol bath.To disrupt the bacteria within the tissue sample 1 ml of TRIzol Reagent(Gibco BRL, Life Technologies) is added followed by enough 0.1 mmzirconia/silica beads to almost fill the tube, the lid is replacedtaking care not to get any beads into the screw thread so as to ensure agood seal and eliminate aerosol generation. The sample is thenhomogenised in a Mini-BeadBeater Type BX-4 (Biospec Products). Necroticfatty tissue is strain treated for 100 seconds at 5000 rpm in order toachieve bacterial lysis. In vivo grown bacteria require longer treatmentthan in vitro grown Staphylococcus aureus bacteria which are disruptedby a 30 second bead-beat.

After bead-beating, the tubes are chilled on ice before opening in afume-hood as heat generated during disruption may degrade the TRIzol andrelease cyanide. 200 μl of chloroform is then added and the tubes shakenby hand for 15 seconds to ensure complete mixing. After 2-3 minutes atroom temperature the tubes are spun down at 12,000×g, 4° C. for 15minutes and RNA extraction is then continued according to the methodgiven by the manufacturers of TRIzol Reagent, i.e. the aqueous phase,approx 0.6 ml, is transferred to a sterile Eppendorf tube and 0.5 ml ofisopropanol is added. After 10 minutes at room temperature, the samplesare spun at 12,000×g, 4° C. for 10 minutes. The supernatant is removedand discarded, then the RNA pellet is washed with 1 ml 75% ethanol. Abrief vortex is used to mix the sample before centrifuging at 7,500×g,4° C. for 5 minutes. The ethanol is removed and the RNA pellet driedunder vacuum for no more than 5 minutes. Samples are then resuspended byrepeated pipetting in 100 μl of DEPC treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at -80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at -20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1× TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue, 1× MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a ³² Plabelled oligonucletide probe specific to 16s rRNA of Staphylococcusaureus (K. Greisen, M. Loeffelholz, A. Purohit and D. Leong. J.Clin.(1994) Microbiol. 32 335-351 ). An oligonucleotide of thesequence:5'-gctcctaaaaggttactccaccggc-3' SEQ ID NO:7! is used as aprobe. The size of the hybridising band is compared to that of controlRNA isolated from in vitro grown Staphylococcus aureus WCUH29 in theNorthern blot. Correct sized bacterial 16s rRNA bands can be detected intotal RNA samples which show extensive degradation of the mammalian RNAwhen visualised on TBE gels.

d) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA:

DNA was removed from 73 μl samples of RNA by a 15 minute treatment onice with 3 units of DNAaseI, amplification grade (Gibco BRL, LifeTechnologies) in the buffer supplied with the addition of 200 units ofRnasin (Promega) in a final volume of 90 μl.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 73 μl of DEPC treatedwater with the addition of Rnasin, as described in Method 1.

e) The preparation of cDNA from RNA samples derived from infectedtissue:

10 μl samples of DNAase treated RNA are reverse transcribed using aSuperScript Preamplification System for First Strand cDNA Synthesis kit(Gibco BRL, Life Technologies) according to the manufacturersinstructions. A 1 nanogram aliquot of random hexamers is used to primeeach reaction. Controls without the addition of SuperScriptII reversetranscriptase are also run. Both +/-RT samples are treated with RNaseHbefore proceeding to the PCR reaction.

f) The use of PCR to determine the presence of a bacterial cDNA species:

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 45 μl PCR SUPERMIX (Gibco BRL, Life Technologies); 1 μl 50mM MgCl₂, to adjust final concentration to 2.5 mM; 1 μl PCR primers(optimally 18-25 basepairs in length and designed to possess similarannealing temperatures), each primer at 10 mM initial concentration; and2 μl cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 5 minutes at 95° C., then 50 cycles of 30 seconds each at 94°C., 42° C. and 72° C. followed by 3 minutes at 72° C. and then a holdtemperature of4° C. The number of cycles is optimally 30-50 to determinethe appearance or lack of a PCR product, and optimally 8-30 cycles if anestimation of the starting quantity of cDNA from the RT reaction is tobe made; 10 μl aliquots are then run out on 1% 1×TBE gels stained withethidium bromide with PCR product, if present, sizes estimated bycomparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies).Alternatively if the PCR products are conveniently labelled by the useof a labelled PCR primer (e.g. labelled at the 5' end with a dye) asuitable aliquot of the PCR product is run out on a polyacrylamidesequencing gel and its presence and quantity detected using a suitablegel scanning system (e.g. ABI Prism™ 377 Sequencer using GeneScan ™software as supplied by Perkin Elmer).

RT/PCR controls may include +/- reverse transcriptase reactions, 16srRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Staphylococcus aureus WCUH29 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStaphylococcus aureus WCUH29 total DNA. PCR reactions are set up and runas described above using approx. 1 μg of DNA in place of the cDNA and 35cycles of PCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: (1) Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and (2) Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in -RT controls.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 7    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1842 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATGAGTCAATTCCTCACACAATTGAAAGATAATGTTTTAGTAGCTGATGGCGCTATTGGA60    ACCATTTTATACTCTGAAGGATTAGACACCTGTCCAGAAGCATATAATCTTAGCCATCCA120    GATAAAGTTGAACGCATCCATCGTTCATATATTGAAGCCGGTGCTGATGTCATTCAAACC180    AATACTTATGGTGCAAATTTTGAAAAGTTAAAACGATTCGGTCTTGAAGATAAAGTTAAA240    GCAATACATCAAGCCGCCGTTCGCATCGCAAAAAAAGCAGCAAATAAAGATACGTATATA300    TTAGGCACAGTTGGTGGGTTTAGAGGTATCAAACAAGAGGATATCAGCTTACAAACTATT360    CTTTATCATACTGAAATTCAAATAGACACCTTAATTGAAGAAGGCGTTGACGCGCTACTT420    TTCGAAACGTATTACGACCTAGAAGAGTTAACAAATGTCATTTCACGAACGAGAAAGAAA480    TACGACATTCCAATCATTGCTCAATTAACCGCTTCAAACACAAATTACTTAGTTAATGGT540    CAGGCAATCAATGAAGGATTAAAACAACTCGTTCAATGTGGTGCAAACATCGTGGGACTC600    AATTGTCATCATGGTCCGCACCATATGCAAGAGTCTTTCACACATATTGAATTACCAGAG660    CACGCATTCTTATCTTGTTATCCAAATGCCAGCTTATTAGATATTGAAAATAGTGAATTT720    AAGTATAGTGACAATGCACAATATTTCGGTCAAGTTGCTCAAAATCTAATTCGCGAAGGT780    GTTCGTTTAATTGGTGGTTGCTGTGGTACAACGCCAGAGCACATCAAATTTATTAAAGAA840    TCTATTCAGACACTTAAACCTGTTAATGACAAAAAAGTGATTCCGATACCAACGAAAGCA900    CTTTTCAATCCATCTCAAAATAAAGTTAGACAATCATTAACATCTAAGGTTCAAGAACGT960    CCAACCGTTATTATCGAATTGGATACACCGAAACATTTAGACACGGATAGATTTTTTGAA1020    AATATCGCTAAACTTGATAAAGCTAATGTAGATGCGGTAACACTCGCAGATAATTCATTG1080    GCAACTGTCAGAATTAGCAATATTGCTGCTGCTAGCTTAATTAAGCAATATTACAATATT1140    GAACCACTCGTACATATTACATGTCGAGACCGAAACTTAATCGGCTTGCAGTCCCATTTA1200    CTTGGATTATCGCTCATTGGCGTTAACGAAATATTAGCCATAACTGGTGATCCTTCAAAA1260    GTTGGTCACTTACCAGGTGCAACCAATGTCTATGATGTTAATTCTAAAGGATTAACTGAA1320    CTCGCTCTAAGATTTAATCAAGGTATTAACACTGACGGTGATGCGCTGAAGAAACGTACA1380    CACTTCAACATCGCTGGCGCCTTTAACCCTAATGTTCGTAAATTAGATGGTGCCGTCAAA1440    AGATTAGAGAAAAAGATAGAAAGCGGAATGTCTTATTTTATAACACAACCCGTGTACAGC1500    AAAGAGAAAATCATTGAAATTTACCATGCCACTAAGCACTTGAACAAACCATTTTTCATA1560    GGCATTATGCCTATCGCAAGTTACAAAAACGCACTCTTTTTGCATAATGAAGTGCCAGGA1620    ATCAAGATGTCAGATGAAATTTTACAACAATTTGAAGCAGTTAAAGATGATAAAGCCAAA1680    ACACGAGAACTAAGTCTTAAGCTTTCAAAGGATTTAATCGATACTGTTCATGAATATTTT1740    AATGGTTTATACATTATCACACCGTTTCAAAATGTCGAAGATTCATTAGAACTTGCAGCA1800    TACTCAAAATCTATTACTGCTCACAAGGAGGCAATATTATGA1842    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 613 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetSerGlnPheLeuThrGlnLeuLysAspAsnValLeuValAlaAsp    151015    GlyAlaIleGlyThrIleLeuTyrSerGluGlyLeuAspThrCysPro    202530    GluAlaTyrAsnLeuSerHisProAspLysValGluArgIleHisArg    354045    SerTyrIleGluAlaGlyAlaAspValIleGlnThrAsnThrTyrGly    505560    AlaAsnPheGluLysLeuLysArgPheGlyLeuGluAspLysValLys    65707580    AlaIleHisGlnAlaAlaValArgIleAlaLysLysAlaAlaAsnLys    859095    AspThrTyrIleLeuGlyThrValGlyGlyPheArgGlyIleLysGln    100105110    GluAspIleSerLeuGlnThrIleLeuTyrHisThrGluIleGlnIle    115120125    AspThrLeuIleGluGluGlyValAspAlaLeuLeuPheGluThrTyr    130135140    TyrAspLeuGluGluLeuThrAsnValIleSerArgThrArgLysLys    145150155160    TyrAspIleProIleIleAlaGlnLeuThrAlaSerAsnThrAsnTyr    165170175    LeuValAsnGlyGlnAlaIleAsnGluGlyLeuLysGlnLeuValGln    180185190    CysGlyAlaAsnIleValGlyLeuAsnCysHisHisGlyProHisHis    195200205    MetGlnGluSerPheThrHisIleGluLeuProGluHisAlaPheLeu    210215220    SerCysTyrProAsnAlaSerLeuLeuAspIleGluAsnSerGluPhe    225230235240    LysTyrSerAspAsnAlaGlnTyrPheGlyGlnValAlaGlnAsnLeu    245250255    IleArgGluGlyValArgLeuIleGlyGlyCysCysGlyThrThrPro    260265270    GluHisIleLysPheIleLysGluSerIleGlnThrLeuLysProVal    275280285    AsnAspLysLysValIleProIleProThrLysAlaLeuPheAsnPro    290295300    SerGlnAsnLysValArgGlnSerLeuThrSerLysValGlnGluArg    305310315320    ProThrValIleIleGluLeuAspThrProLysHisLeuAspThrAsp    325330335    ArgPhePheGluAsnIleAlaLysLeuAspLysAlaAsnValAspAla    340345350    ValThrLeuAlaAspAsnSerLeuAlaThrValArgIleSerAsnIle    355360365    AlaAlaAlaSerLeuIleLysGlnTyrTyrAsnIleGluProLeuVal    370375380    HisIleThrCysArgAspArgAsnLeuIleGlyLeuGlnSerHisLeu    385390395400    LeuGlyLeuSerLeuIleGlyValAsnGluIleLeuAlaIleThrGly    405410415    AspProSerLysValGlyHisLeuProGlyAlaThrAsnValTyrAsp    420425430    ValAsnSerLysGlyLeuThrGluLeuAlaLeuArgPheAsnGlnGly    435440445    IleAsnThrAspGlyAspAlaLeuLysLysArgThrHisPheAsnIle    450455460    AlaGlyAlaPheAsnProAsnValArgLysLeuAspGlyAlaValLys    465470475480    ArgLeuGluLysLysIleGluSerGlyMetSerTyrPheIleThrGln    485490495    ProValTyrSerLysGluLysIleIleGluIleTyrHisAlaThrLys    500505510    HisLeuAsnLysProPhePheIleGlyIleMetProIleAlaSerTyr    515520525    LysAsnAlaLeuPheLeuHisAsnGluValProGlyIleLysMetSer    530535540    AspGluIleLeuGlnGlnPheGluAlaValLysAspAspLysAlaLys    545550555560    ThrArgGluLeuSerLeuLysLeuSerLysAspLeuIleAspThrVal    565570575    HisGluTyrPheAsnGlyLeuTyrIleIleThrProPheGlnAsnVal    580585590    GluAspSerLeuGluLeuAlaAlaTyrSerLysSerIleThrAlaHis    595600605    LysGluAlaIleLeu    610    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1839 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ATGAGTCAATTCCTCACACAATTGAAAGATAATGTTTTAGTAGCTGATGGCGCTATTGGA60    ACCATTTTATACTCTGAAGGATTAGACACCTGTCCAGAAGCATATAATCTTAGCCATCCA120    GATAAAGTTGAACGCATCCATCGTTCATATATTGAAGCCGGTGCTGATGTCATTCAAACC180    AATACTTATGGTGCAAATTTTGAAAAGTTAAAACGATTCGGTCTTGAAGATAAAGTTAAA240    GCAATACATCAAGCCGCCGTTCGCATCGCAAAAAAAGCAGCAAATAAAGATACGTATATA300    TTAGGCACAGTTGGTGGGTTTAGAGGTATCAAACAAGAGGATATCAGCTTACAAACTATT360    CTTTATCATACTGAAATTCAAATAGACACCTTAATTGAAGAAGGCGTTGACGCGCTACTT420    TTCGAAACGTATTACGACCTAGAAGAGTTAACAAATGTCATTTCACGAACGAGAAAGAAA480    TACGACATTCCAATCATTGCTCAATTAACCGCTTCAAACACAAATTACTTAGTTAATGGT540    CAGGCAATCAATGAAGGATTAAAACAACTCGTTCAATGTGGTGCAAACATCGTGGGACTC600    AATTGTCATCATGGTCCGCACCATATGCAAGAGTCTTTCACACATATTGAATTACCAGAG660    CACGCATTCTTATCTTGTTATCCAAATGCCAGCTTATTAGATATTGAAAATAGTGAATTT720    AAGTATAGTGACAATGCACAATATTTCGGTCAAGTTGCTCAAAATCTAATTCGCGAAGGT780    GTTCGTTTAATTGGTGGTTGCTGTGGTACAACGCCAGAGCACATCAAATTTATTAAAGAA840    TCTATTCAGACACTTAAACCTGTTAATGACAAAAAAGTGATTCCGATACCAACGAAAGCA900    CTTTTCAATCCATCTCAAAATAAAGTTAGACAATCATTAACATCTAAGGTTCAAGAACGT960    CCAACCGTTATTATCGAATTGGATACACCGAAACATTTAGACACGGATAGATTTTTTGAA1020    AATATCGCTAAACTTGATAAAGCTAATGTAGATGCGGTAACACTCGCAGATAATTCATTG1080    GCAACTGTCAGAATTAGCAATATTGCTGCTGCTAGCTTAATTAAGCAATATTACAATATT1140    GAACCACTCGTACATATTACATGTCGAGACCGAAACTTAATCGGCTTGCAGTCCCATTTA1200    CTTGGATTATCGCTCATTGGCGTTAACGAAATATTAGCCATAACTGGTGATCCTTCAAAA1260    GTTGGTCACTTACCAGGTGCAACCAATGTCTATGATGTTAATTCTAAAGGATTAACTGAA1320    CTCGCTCTAAGATTTAATCAAGGTATTAACACTGACGGTGATGCGCTGAAGAAACGTACA1380    CACTTCAACATCGCTGGCGCCTTTAACCCTAATGTTCGTAAATTAGATGGTGCCGTCAAA1440    AGATTAGAGAAAAAGATAGAAAGCGGAATGTCTTATTTTATAACACAACCCGTGTACAGC1500    AAAGAGAAAATCATTGAAATTTACCATGCCACTAAGCACTTGAACAAACCATTTTTCATA1560    GGCATTATGCCTATCGCAAGTTACAAAAACGCACTCTTTTTGCATAATGAAGTGCCAGGA1620    ATCAAGATGTCAGATGAAATTTTACAACAATTTGAAGCAGTTAAAGATGATAAAGCCAAA1680    ACACGAGAACTAAGTCTTAAGCTTTCAAAGGATTTAATCGATACTGTTCATGAATATTTT1740    AATGGTTTATACATTATCACACCGTTTCAAAATGTCGAAGATTCATTAGAACTTGCAGCA1800    TACTCAAAATCTATTACTGCTCACAAGGAGGCAATATTA1839    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 613 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetSerGlnPheLeuThrGlnLeuLysAspAsnValLeuValAlaAsp    151015    GlyAlaIleGlyThrIleLeuTyrSerGluGlyLeuAspThrCysPro    202530    GluAlaTyrAsnLeuSerHisProAspLysValGluArgIleHisArg    354045    SerTyrIleGluAlaGlyAlaAspValIleGlnThrAsnThrTyrGly    505560    AlaAsnPheGluLysLeuLysArgPheGlyLeuGluAspLysValLys    65707580    AlaIleHisGlnAlaAlaValArgIleAlaLysLysAlaAlaAsnLys    859095    AspThrTyrIleLeuGlyThrValGlyGlyPheArgGlyIleLysGln    100105110    GluAspIleSerLeuGlnThrIleLeuTyrHisThrGluIleGlnIle    115120125    AspThrLeuIleGluGluGlyValAspAlaLeuLeuPheGluThrTyr    130135140    TyrAspLeuGluGluLeuThrAsnValIleSerArgThrArgLysLys    145150155160    TyrAspIleProIleIleAlaGlnLeuThrAlaSerAsnThrAsnTyr    165170175    LeuValAsnGlyGlnAlaIleAsnGluGlyLeuLysGlnLeuValGln    180185190    CysGlyAlaAsnIleValGlyLeuAsnCysHisHisGlyProHisHis    195200205    MetGlnGluSerPheThrHisIleGluLeuProGluHisAlaPheLeu    210215220    SerCysTyrProAsnAlaSerLeuLeuAspIleGluAsnSerGluPhe    225230235240    LysTyrSerAspAsnAlaGlnTyrPheGlyGlnValAlaGlnAsnLeu    245250255    IleArgGluGlyValArgLeuIleGlyGlyCysCysGlyThrThrPro    260265270    GluHisIleLysPheIleLysGluSerIleGlnThrLeuLysProVal    275280285    AsnAspLysLysValIleProIleProThrLysAlaLeuPheAsnPro    290295300    SerGlnAsnLysValArgGlnSerLeuThrSerLysValGlnGluArg    305310315320    ProThrValIleIleGluLeuAspThrProLysHisLeuAspThrAsp    325330335    ArgPhePheGluAsnIleAlaLysLeuAspLysAlaAsnValAspAla    340345350    ValThrLeuAlaAspAsnSerLeuAlaThrValArgIleSerAsnIle    355360365    AlaAlaAlaSerLeuIleLysGlnTyrTyrAsnIleGluProLeuVal    370375380    HisIleThrCysArgAspArgAsnLeuIleGlyLeuGlnSerHisLeu    385390395400    LeuGlyLeuSerLeuIleGlyValAsnGluIleLeuAlaIleThrGly    405410415    AspProSerLysValGlyHisLeuProGlyAlaThrAsnValTyrAsp    420425430    ValAsnSerLysGlyLeuThrGluLeuAlaLeuArgPheAsnGlnGly    435440445    IleAsnThrAspGlyAspAlaLeuLysLysArgThrHisPheAsnIle    450455460    AlaGlyAlaPheAsnProAsnValArgLysLeuAspGlyAlaValLys    465470475480    ArgLeuGluLysLysIleGluSerGlyMetSerTyrPheIleThrGln    485490495    ProValTyrSerLysGluLysIleIleGluIleTyrHisAlaThrLys    500505510    HisLeuAsnLysProPhePheIleGlyIleMetProIleAlaSerTyr    515520525    LysAsnAlaLeuPheLeuHisAsnGluValProGlyIleLysMetSer    530535540    AspGluIleLeuGlnGlnPheGluAlaValLysAspAspLysAlaLys    545550555560    ThrArgGluLeuSerLeuLysLeuSerLysAspLeuIleAspThrVal    565570575    HisGluTyrPheAsnGlyLeuTyrIleIleThrProPheGlnAsnVal    580585590    GluAspSerLeuGluLeuAlaAlaTyrSerLysSerIleThrAlaHis    595600605    LysGluAlaIleLeu    610    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CCTCACACAATTGAAAGATAATG23    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GTGAAAGACTCTTGCATATGG21    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GCTCCTAAAAGGTTACTCCACCGGC25    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding SEQID NO:
 2. 2. An isolated nucleic acid segment comprising a nucleotidesequence which is fully complementary to the polynucleotide segment ofclaim
 1. 3. An isolated vector comprising the polynucleotide segment ofclaim
 1. 4. An isolated vector comprising the nucleic acid segment ofclaim
 2. 5. An isolated host cell comprising the vector of claim
 3. 6.An isolated host cell comprising the vector of claim
 4. 7. A process forproducing a polypeptide encoded by said polynucleotide segmentcomprising culturing the host cell of claim 5 under conditionssufficient for the production of said polypeptide.
 8. An isolatedpolynucleotide segment comprising a nucleotide sequence comprising SEQID NO:
 1. 9. An isolated nucleic acid segment comprising a nucleotidesequence which is fully complementary to the polynucleotide segment ofclaim
 8. 10. An isolated vector comprising the polynucleotide segment ofclaim
 8. 11. An isolated vector comprising the nucleic acid segment ofclaim
 9. 12. An isolated host cell comprising the vector of claim 10.13. An isolated host cell comprising the vector of claim
 11. 14. Aprocess for producing a yitJ polypeptide comprising culturing the hostcell of claim 12 under conditions sufficient for the production of saidpolypeptide.